Fuel ethanol is currently produced from feedstocks such as corn starch, sugar cane, and sugar beets. However, the production of ethanol from these feedstocks suffers from the limitation that much of the farmland which is suitable for their production is already in use as a food source for humans and animals. A further disadvantage of the use of these feedstocks is that fossil fuels are used in the conversion processes. Thus, these processes have only a limited impact on reducing greenhouse gases.
The production of ethanol from lignocellulose-containing feedstocks, such as agricultural wastes and forestry wastes, has received much attention in recent years due their low cost and wide availability. In addition, agricultural and forestry wastes are typically burned or land-filled, and thus using these lignocellulosic feedstocks for ethanol production offers an attractive alternative for disposing of them. Yet another advantage of these feedstocks is that a byproduct known as lignin, which remains after the cellulose conversion process, can be used as a fuel to power the process instead of fossil fuels. Several studies have concluded that, when the entire production and consumption cycle is taken into account, the use of ethanol produced from cellulose generates close to nil greenhouse gases.
Lignocellulosic materials have also been considered for producing other fermentation products besides ethanol. Examples of such products include lactic acid, sorbitol, acetic acid, citric acid, ascorbic acid, propanediol, butanediol, xylitol, acetone, and butanol.
The first chemical processing step for converting lignocellulosic feedstock to ethanol, or other fermentation products, involves breaking down the fibrous lignocellulosic material to liberate sugar monomers, such as glucose and xylose, from the feedstock for conversion to ethanol in a subsequent step of fermentation. The two primary processes to break down the fibrous material are acid or alkali hydrolysis, which involve the hydrolysis of the feedstock using a single step of chemical treatment, and enzymatic hydrolysis, which involves an acid or alkali pretreatment followed by hydrolysis with cellulase enzymes.
In acid or alkali hydrolysis, the lignocellulosic feedstock is contacted with a strong acid or alkali under conditions sufficient to hydrolyze both the cellulose and hemicellulose components of the feedstock to their monomeric constituents.
In the enzymatic hydrolysis process, the lignocellulosic feedstock is first subjected to a pretreatment under conditions which are similar to but milder than those in the acid or alkali hydrolysis process. The purpose of the pretreatment is to increase the cellulose surface area and convert the fibrous feedstock to a muddy texture, with limited conversion of the cellulose to glucose. If the pretreatment is conducted with acid, the hemicellulose component of the feedstock is hydrolyzed to xylose, arabinose, galactose and mannose, while alkali pretreatment does not hydrolyze sugar polymers, but rather opens up the surface of the substrate by reacting with acidic groups present on the hemicellulose. After pretreatment, the cellulose is hydrolyzed to glucose in a subsequent step that employs cellulase enzymes.
Cellulase is a generic term denoting a multi-enzyme mixture comprising exo-cellobiohydrolases (CBH), endoglucanases (EG), β-glucosidase, xylanases and β-xylosidase. The CBH and EG enzymes catalyze the hydrolysis of the cellulose (β1,4-D-glucan linkages). The CBH enzymes, CBHI and CBHII, act on the ends of the glucose polymers in cellulose microfibrils and liberate cellobiose, while the EG enzymes act at random locations on the cellulose. Together, cellulase enzymes hydrolyze cellulose to cellobiose, which, in turn, is hydrolyzed to glucose by β-glucosidase. The xylanase enzymes, such as xylanase 1 (Xyn1), xylanase 2 (Xyn2) and β-xylosidase, are typically present in the cellulase enzyme mixture and hydrolyze any xylan present in the feedstock. The presence of such enzymes may be advantageous in cases where significant amounts of xylan are present in the pretreated feedstock.
The next step of the process involves subjecting the sugar stream to fermentation to produce ethanol or other fermentation products. If glucose is the predominant substrate present, the fermentation is typically carried out with a Saccharomyces spp. yeast which converts this sugar and other hexose sugars present to ethanol. Furthermore, the pentose sugar, xylose, which arises from acidic pretreatment, can be fermented to ethanol, although wild-type Saccharomyces strains do not naturally contain the genes required for converting xylose to ethanol, and thus must be introduced into the yeast to allow for this conversion. Recombinant yeasts that are able to convert xylose to ethanol are described, for example, in U.S. Pat. Nos. 5,789,210, 6,475,768 and European Patent No. EP 1 727 890 B1.
One problem with the fermentation of sugar to ethanol, particularly the fermentation of xylose to ethanol, is that the efficiency of the process is limited by the slow kinetics of the reaction. In order to increase the volumetric rate of conversion of xylose to ethanol, it is known to increase the concentration of yeast in the fermentor. This may be carried out by continuously separating yeast from the harvested fermentation broth, such as by centrifugation, and then re-circulating the yeast back to the fermentor. The ethanol in the liquid resulting from the separation of the yeast is recovered by distillation or other known techniques. By re-introducing yeast back to the reactor in this manner, the concentration of yeast in the fermentor is continuously increased, which, in turn, substantially increases the volumetric productivity of the fermentation.
However, a problem with repeated re-circulation of yeast is that microbes, such as bacteria, are also recycled along with the yeast. This can lead to increased levels of microbial contaminants and can result in the production of unwanted fermentation products such as lactic acid. The presence of microbial contaminants producing lactic acid decreases the yield of the desired fermentation product, as well as result in a product of low purity.
A known method for disinfecting yeast-containing solutions involves washing the yeast with acid solutions, typically phosphoric or sulfuric acid, to lower the pH to below about 2.5. The goal of this treatment is to destroy contaminating microorganisms which cannot withstand low pH conditions, without a substantial reduction in yeast viability or fermentative capacity. However, washing yeast under such harshly acidic conditions subjects them to significant stress, which can reduce both viability and fermentative capacity and thus the yield of ethanol produced. Furthermore, acid washing is ineffective at destroying acid-tolerant bacteria, such as lactic acid producing bacteria.
Chang et al. (Appl. Environ. Microbiol. 63: 1-6, 1997) disclose the use of sulfite and hydrogen peroxide to control bacterial contamination in a fermentation process producing ethanol from malt extract. The sulfite treatment is performed on yeast cells removed from the fermentor and concentrated by filtration. However, bacterial contaminants (two Lactobacillus strains) normally found in industrial Saccharomyces fermentations were susceptible to sulfite only in the presence of oxygen. The addition of oxygen to the concentrated yeast slurry is not beneficial to the subsequent fermentation process, which is anaerobic. The yeast cells would therefore need to be reconditioned to anaerobic conditions in order to increase the efficiency of ethanol fermentation process. Other results presented demonstrate that hydrogen peroxide was effective in reducing the viability of one of the Lactobacillus species tested.
U.S. Publication Nos. 2003/0190742 and 2002/0117445 (Whiteman) disclose the sterilization of a fermentation tank between the growth of different fermentation batches by treatment with water or chlorine dioxide, followed by exposure to UV light or the addition of an acidic solution of pH 2. However, there is no disclosure of treating a yeast-containing solution to control the propagation of microbial contaminants.
British Patent No. 366,525 (Wadsworth and Wickenden) discloses the sterilization of raw sugar melt and syrups obtained from sugar cane for subsequent transportation. The sterilization involves subjecting the sugar to a weak solution of chlorine gas or liquid chlorine itself, which destroys residue of invertase, yeast and bacteria, and renders the impurities present more amenable to treatment for their subsequent removal. However, the treatment is carried out to destroy both yeast and bacteria, and thus would not be an effective method of controlling bacterial contamination in a yeast culture.
U.S. Pat. No. 7,078,201 (Burmaster) discloses a method of monitoring and controlling the oxidation-reduction potential during a fermentation of corn mash to ethanol. By raising the oxidation-reduction potential, glycerol formation is lowered, which, in turn, increases the ethanol yield. Oxidants which may be utilized for this purpose include ozone, dihalides (chlorine, bromine and iodine), chlorine dioxide, potassium permanganate and air or oxygen sparging. However, the disclosure is directed to maximizing ethanol yield by reducing glycerol production and does not address the need to control the propagation of unwanted microbes.
U.S. Publication No. 2006/0251762 (Jansen et al.) discloses that it is known to use sodium hypochlorite or chlorine dioxide to control the propagation of micro-organisms during the production of ethanol from wheat. However, the use of these oxidizing chemicals is undesirable since it can affect the “functional visco-elastic properties of the vital wheat gluten”, thus reducing its quality. Furthermore, Jansen et al. teaches production of ethanol from wheat, which is a starch-containing feedstock. In starch-conversion processes, yeast recycling is not employed since conversion the fermented solutions arising from these processes contain solids and the conversion of glucose to ethanol is generally not limited by slow kinetics.
Johnson and Kunz (The New Brewer, 1998, Coming Clean—A New Method of Washing Yeast Using Chlorine Dioxide Vol. 15 #5-P56) disclose the addition of chlorine dioxide to a yeast slurry during the brewing of beer. However, there is no disclosure of producing a fermentation product from a lignocellulosic feedstock comprising hemicellulose, or any mention of the specific problems encountered when converting xylose to ethanol or other fermentation products.
PCT Publication WO 2007/149450 discloses a method for preventing the growth of bacterial contaminants in yeast fermentations via the addition of stabilized chlorine dioxide to a yeast fermentation system. Although the chlorine dioxide may be added with the inoculant, or fermentable sugars prior to their addition to the fermentation system, the method is directed to the prevention of the growth of bacteria during the fermentation reaction. The method does not provide a solution for reducing bacterial contaminants that may accumulate during the yeast fermentation reaction or a solution for concentrating the yeast cells in the fermentation system to improve the efficiency of the process for fermenting hemicellulose-derived hydrolysates.
PCT Publication WO 2007/097874 also discloses a method for reducing bacterial contamination in a yeast fermentation system. In this process, chlorine dioxide is added to the fermentation system, to the fermentable carbohydrate, or to the propagation or conditioning systems used to prepare the inoculum for the fermentation. The method does not provide a solution for reducing bacterial contaminants that may accumulate during the yeast fermentation reaction or a solution for concentrating the yeast cells in the fermentation system to improve the efficiency of the process for fermenting hemicellulose-derived hydrolysates.
At present, there is much difficulty in the art to operate an efficient process for fermenting sugars obtained from the hydrolysis of hemicellulose-containing feedstocks to produce a high yield of a fermentation product. The development of an efficient process remains a critical requirement to convert such feedstocks to fermentation products, such as ethanol.